Small-scale metapopulation structure of a limnophilic fish species in a natural river system investigated using microsatellite genotyping by amplicon sequencing (SSR-GBAS).

Habitat niches of fish species can exert a strong influence on population structure, even on a small geographical scale. In this scope, Pelasgus thesproticus is a great model species to study connectivity in riverine environments owing to its naturally patchy habitat distribution. Furthermore, it is important to conduct such studies in near-natural systems to avoid the impact of human disturbances on the river, such as fragmentation, morphological changes and habitat degradation. In this sense, the Vjosa in Albania is an excellent study area. A total of 204 individuals were sampled from five locations in the lower Vjosa and two tributaries and genotyped with 33 newly designed microsatellites loci using high throughput sequencing. The application of microsatellite genotyping by sequencing revealed genetic structure and some differentiation, even at a small spatial scale (< 65 river km). A total of 500 alleles were found with an average of 0.93 private alleles among sites with rather low FST values (< 0.04). The extent of admixture observed in some populations indicate that the genetic structure is mainly influenced by upstream populations, either from the main river itself or from tributaries. In addition, the connection between a tributary and the other sites is disrupted by the flow regime, which is reflected in a high degree of divergence from the other populations. Our results indicate that hydrological conditions of the flowing river present strong barriers to gene flow, particularly in the upstream direction, but at the same time act as dispersal corridors in the downstream direction and exhibit source-sink dynamics in which upstream populations contribute disproportionately to downstream populations for this habitat specialist along the river. It is suggested that processes of colonization and reinforcement may play an important role in shaping the genetic structure of patchily distributed fish species in natural river systems. Future studies should increase the knowledge of dispersal factors, habitat heterogeneity, consequence of source-sink dynamics, and gene flow within the system, which will help to understand and maintain important processes related to metapopulation theory and the potential evolutionary consequences of habitat loss and fragmentation.

The recovery of European freshwater biodiversity has come to a halt.

Owing to a long history of anthropogenic pressures, freshwater ecosystems are among the most vulnerable to biodiversity loss1. Mitigation measures, including wastewater treatment and hydromorphological restoration, have aimed to improve environmental quality and foster the recovery of freshwater biodiversity2. Here, using 1,816 time series of freshwater invertebrate communities collected across 22 European countries between 1968 and 2020, we quantified temporal trends in taxonomic and functional diversity and their responses to environmental pressures and gradients. We observed overall increases in taxon richness (0.73% per year), functional richness (2.4% per year) and abundance (1.17% per year). However, these increases primarily occurred before the 2010s, and have since plateaued. Freshwater communities downstream of dams, urban areas and cropland were less likely to experience recovery. Communities at sites with faster rates of warming had fewer gains in taxon richness, functional richness and abundance. Although biodiversity gains in the 1990s and 2000s probably reflect the effectiveness of water-quality improvements and restoration projects, the decelerating trajectory in the 2010s suggests that the current measures offer diminishing returns. Given new and persistent pressures on freshwater ecosystems, including emerging pollutants, climate change and the spread of invasive species, we call for additional mitigation to revive the recovery of freshwater biodiversity.

Notes on Leuctra signifera Kempny, 1899 and Leuctra austriaca Aubert, 1954 (Plecoptera: Leuctridae), with the description of a new species.

Leuctra signifera Kempny, 1899 and Leuctra austriaca Aubert, 1954 are re-described and their taxonomical status is discussed. New country records are reported for L. austriaca from Hungary and L. signifera from Italy, Slovenia, and Hungary. A new species, Leuctra papukensis sp. n., from Croatia, is described and illustrated. The subspecies Leuctra signifera jahorinensis Kacanski, 1972 is raised to species level.

New records for the Western Balkans cranefly fauna (Diptera, Tipuloidea) with the description of a new Baeoura Alexander (Diptera, Limoniidae).

The cranefly (Tipuloidea) fauna of the Western Balkans is still poorly known. In this study, occurrence data of 77 species is reported, of which two species are newly recorded for Albania, eight species for Bosnia and Herzegovina, twelve for Croatia, and seven for Slovenia, respectively. A new species, Baeouraneretvaensis Kolcsár & d'Oliveira, sp. nov. is described from Bosnia and Herzegovina, Montenegro, and Slovenia. Images of the habitus, wing, and male and female terminalia of the new species are provided. Furthermore, images of male terminalia and wings of thirteen additional species are presented.

Invasion impacts and dynamics of a European-wide introduced species.

Globalization has led to the introduction of thousands of alien species worldwide. With growing impacts by invasive species, understanding the invasion process remains critical for predicting adverse effects and informing efficient management. Theoretically, invasion dynamics have been assumed to follow an "invasion curve" (S-shaped curve of available area invaded over time), but this dynamic has lacked empirical testing using large-scale data and neglects to consider invader abundances. We propose an "impact curve" describing the impacts generated by invasive species over time based on cumulative abundances. To test this curve's large-scale applicability, we used the data-rich New Zealand mud snail Potamopyrgus antipodarum, one of the most damaging freshwater invaders that has invaded almost all of Europe. Using long-term (1979-2020) abundance and environmental data collected across 306 European sites, we observed that P. antipodarum abundance generally increased through time, with slower population growth at higher latitudes and with lower runoff depth. Fifty-nine percent of these populations followed the impact curve, characterized by first occurrence, exponential growth, then long-term saturation. This behaviour is consistent with boom-bust dynamics, as saturation occurs due to a rapid decline in abundance over time. Across sites, we estimated that impact peaked approximately two decades after first detection, but the rate of progression along the invasion process was influenced by local abiotic conditions. The S-shaped impact curve may be common among many invasive species that undergo complex invasion dynamics. This provides a potentially unifying approach to advance understanding of large-scale invasion dynamics and could inform timely management actions to mitigate impacts on ecosystems and economies.

Microbial faecal pollution of river water in a watershed of tropical Ethiopian highlands is driven by diffuse pollution sources.

Tropical communities in the developing world depend heavily on riverine systems for their socioeconomic development. However, these resources are poorly protected from diffuse pollution, and there is a lack of quantitative information regarding the microbial pollution characteristics of riverine water, despite frequently reported gastrointestinal diseases. The aim of our study was to apply faecal taxation (i.e., faecal pellet counting in representative test areas to estimate the potential availability of diffuse pollution sources) in combination with a detailed microbiological faecal pollution analysis in a riverine environment to elucidate the importance of diffuse pollution. To realize this approach, ambient faecal pellets, a multiparametric data set for standard faecal indicator bacteria (SFIB), including Escherichia coli, Clostridium perfringens spores and enterococci from catchment soil and river water, and a number of riverine water physicochemical variables were analysed during a one-year cycle. We demonstrated that the abundance of ambient faecal pellets, which were consistently counted at reference sites in the catchment, was associated with faecal pollution in the river water. Water SFIB, dissolved oxygen, nutrients, conductivity and total suspended solids were strongly linked with the abundance of ambient faecal pellets in the river catchment, as demonstrated by principal component analysis (PCA). Elevated concentrations of SFIB in the riverine water in the absence of rainfall also suggested the direct input of faecal bacteria into the riverine water by livestock (e.g., during watering) and humans (e.g., during bathing). Statistical analyses further revealed that the microbiological water quality of the investigated riverine water was not influenced by SFIB potentially occurring in the soil. This study demonstrates the importance of diffuse faecal pollution sources as major drivers of the microbiological quality of riverine water in the Ethiopian highlands. In addition, the new successfully applied integrated approach could be very useful for developing predictive models, which would aid in forecasting riverine microbiological quality in tropical developing countries.

The coupled socio-ecohydrological evolution of river systems: Towards an integrative perspective of river systems in the 21st century.

River systems have undergone a massive transformation since the Anthropocene. The natural properties of river systems have been drastically altered and reshaped, limiting the use of management frameworks, their scientific knowledge base and their ability to provide adequate solutions for current problems and those of the future, such as climate change, biodiversity crisis and increased demands for water resources. To address these challenges, a socioecologically driven research agenda for river systems that complements current approaches is needed and proposed. The implementation of the concepts of social metabolism and the colonisation of natural systems into existing concepts can provide a new basis to analyse the coevolutionary coupling of social systems with ecological and hydrological (i.e., 'socio-ecohydrological') systems within rivers. To operationalize this research agenda, we highlight four initial core topics defined as research clusters (RCs) to address specific system properties in an integrative manner. The colonisation of natural systems by social systems is seen as a significant driver of the transformation processes in river systems. These transformation processes are influenced by connectivity (RC 1), which primarily addresses biophysical aspects and governance (RC 2), which focuses on the changes in social systems. The metabolism (RC 3) and vulnerability (RC 4) of the social and natural systems are significant aspects of the coupling of social systems and ecohydrological systems with investments, energy, resources, services and associated risks and impacts. This socio-ecohydrological research agenda complements other recent approaches, such as 'socio-ecological', 'socio-hydrological' or 'socio-geomorphological' systems, by focusing on the coupling of social systems with natural systems in rivers and thus, by viewing the socioeconomic features of river systems as being just as important as their natural characteristics. The proposed research agenda builds on interdisciplinarity and transdisciplinarity and requires the implementation of such programmes into the education of a new generation of river system scientists, managers and engineers who are aware of the transformation processes and the coupling between systems.

The rare and enigmatic mayfly Prosopistoma pennigerum (Müller, 1785): Habitat characteristics, recent records from the Volga (Russia) and Vjosa (Albania) rivers, and a proposal for flagship species status.

Nymphs of all 29 described Prosopistoma species share a conspicuous synapomorphy: a round mesonotal shield, the carapace. They occur in the Palaearctic (nine species), the Oriental (12 species) and the Afrotropic as well as Australian regions (six and two species, respectively). Relatively little is known about their ecology, but past and extant distribution patterns indicate an association with undisturbed conditions. Prosopistoma pennigerum is a rare European mayfly with conspicuous nymphs. Formerly common in large rivers, it has been extirpated from central Europe over the last century.This study evaluated general habitat characteristics and human pressures for historical and current records of this rare species. Prosopistoma pennigerum is currently known from only three European rivers, all with gravel substrates, naturally dynamic discharge regimes, summer-warm water temperatures, and little human pressure.This study showed that nymphs from the Vjosa and upper Volga rivers, two relatively natural watercourses 2,000 km apart, are morphologically indistinguishable, and show no variation across a ca. 600-bp fragment of the mitochondrial cytochrome c oxidase I gene.Flagship species were first designated in the 1980s, when charismatic species with high habitat requirements such as the Bengal tiger or the giant panda, but also invertebrates are used to communicate conservation and protection needs. We propose that Europe's rarest mayfly P. pennigerum, with its unusual nymphs and remaining populations in naturally dynamic river courses, can serve as a flagship species promoting the preservation of ecological integrity in European rivers.

The larva of Chaetopteryx rugulosa Kolenati 1848 (Trichoptera: Limnephilidae), with notes on ecology and zoogeography.

This paper gives a description of the hitherto unknown larva of Chaetopteryx rugulosa Kolenati 1848 (Trichoptera: Limnephilidae). Information on the morphology of the larva is given, and the most important diagnostic features are figured. In the context of the known European Chaetopterygini and Stenophylacini species, the larva of C. rugulosa can be separated from morphologically close species by metanotal sclerite morphology, setation, the extension of the lateral fringe, and by case morphology.

The larva of Plectrocnemia scruposa McLachlan 1880 (Trichoptera, Polycentropodidae.

This paper describes the previously unknown larva of Plectrocnemia scruposa McLachlan 1880. Information on the morphology of the 5th larval instar is given, and the most important diagnostic features are illustrated. In the context of existing identification keys, P. scruposa keys together with P. brevis McLachlan 1871, P. conspersa (Curtis 1834), and P. laetabilis McLachlan 1880. These species can be separated by the length of the outermost seta of the posterolateral trio on the last abdominal tergum and by presence/absence of a central pigmented mark on the posterior margin of the labrum. According to mandible morphology, the larva of this species appears to be mainly a predator and passive filter feeder of particulate organic matter.

A new Drusinae species from the western Alps with comments on the subfamily and an updated key to filtering carnivore larvae of Drusinae species (Insecta: Trichoptera: Limnephilidae).

A new Drusinae species, Drusus katagelastos sp. nov., of the Drusus chapmani Species Complex, is described based on a male and associated larvae. Adult-larval association was achieved through DNA barcoding. The male of the new species differ from that of its congeners in the formation of the intermediate appendages and parameres. Information on the morphology of the larva is given, and important diagnostic features are discussed. In the context of filtering carnivore Drusinae, the larva of the new species can be separated from other filtering carnivore species by the dense cover of long translucent bristles within the frontal cavity surrounded by a circular corona of long bristles. Drusus katagelastos sp. nov. is known from only northwestern Italy (Piemonte).

Impacts of multiple stressors on freshwater biota across spatial scales and ecosystems.

Climate and land-use change drive a suite of stressors that shape ecosystems and interact to yield complex ecological responses (that is, additive, antagonistic and synergistic effects). We know little about the spatial scales relevant for the outcomes of such interactions and little about effect sizes. These knowledge gaps need to be filled to underpin future land management decisions or climate mitigation interventions for protecting and restoring freshwater ecosystems. This study combines data across scales from 33 mesocosm experiments with those from 14 river basins and 22 cross-basin studies in Europe, producing 174 combinations of paired-stressor effects on a biological response variable. Generalized linear models showed that only one of the two stressors had a significant effect in 39% of the analysed cases, 28% of the paired-stressor combinations resulted in additive effects and 33% resulted in interactive (antagonistic, synergistic, opposing or reversal) effects. For lakes, the frequencies of additive and interactive effects were similar for all spatial scales addressed, while for rivers these frequencies increased with scale. Nutrient enrichment was the overriding stressor for lakes, with effects generally exceeding those of secondary stressors. For rivers, the effects of nutrient enrichment were dependent on the specific stressor combination and biological response variable. These results vindicate the traditional focus of lake restoration and management on nutrient stress, while highlighting that river management requires more bespoke management solutions.

Diversity and Ecosystem Services of Trichoptera.

The holometabolous insect order Trichoptera (caddisflies) includes more known species than all of the other primarily aquatic orders of insects combined. They are distributed unevenly; with the greatest number and density occurring in the Oriental Biogeographic Region and the smallest in the East Palearctic. Ecosystem services provided by Trichoptera are also very diverse and include their essential roles in food webs, in biological monitoring of water quality, as food for fish and other predators (many of which are of human concern), and as engineers that stabilize gravel bed sediment. They are especially important in capturing and using a wide variety of nutrients in many forms, transforming them for use by other organisms in freshwaters and surrounding riparian areas. The general pattern of evolution for trichopteran families is becoming clearer as more genes from more taxa are sequenced and as morphological characters are becoming understood in greater detail. This increasingly credible phylogeny provides a foundation for interpreting and hypothesizing the functional traits of this diverse order of freshwater organisms and for understanding the richness of the ecological services corresponding with those traits. Our research also is gaining insight into the timing of evolutionary diversification in the order. Correlations for the use of angiosperm plant material as food and case construction material by the earliest ancestors of infraorder Plenitentoria-by at least 175 Ma-may provide insight into the timing of the origin of angiosperms.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

Reintroduction of freshwater macroinvertebrates: challenges and opportunities.

Species reintroductions - the translocation of individuals to areas in which a species has been extirpated with the aim of re-establishing a self-sustaining population - have become a widespread practice in conservation biology. Reintroduction projects have tended to focus on terrestrial vertebrates and, to a lesser extent, fishes. Much less effort has been devoted to the reintroduction of invertebrates into restored freshwater habitats. Yet, reintroductions may improve restoration outcomes in regions where impoverished regional species pools limit the self-recolonisation of restored freshwaters. We review the available literature on macroinvertebrate reintroductions, focusing on identifying the intrinsic and extrinsic factors that determine their success or failure. Our study reveals that freshwater macroinvertebrate reintroductions remain rare, are often published in the grey literature and, of the attempts made, approximately one-third fail. We identify life-cycle complexity and remaining stressors as the two factors most likely to affect reintroduction success, illustrating the unique challenges of freshwater macroinvertebrate reintroductions. Consideration of these factors by managers during the planning process and proper documentation - even if a project fails - may increase the likelihood of successful outcomes in future reintroduction attempts of freshwater macroinvertebrates.

LaRiMo - A simple and efficient GIS-based approach for large-scale morphological assessment of large European rivers.

Large rivers cover and function over large spatial extents. Accordingly, the detailed assessment of their morphology is complex. Here, a methodology is presented to assess large rivers' morphology (LaRiMo) based on free datasets of geographic information systems. This approach could help to achieve a comparable, transboundary assessment of large river morphology to support the implementation of the European Water Framework Directive. The analyses are based on seven parameters describing processes and characteristics related to large river morphology. These parameters are evaluated for and compared between the rivers Danube, Elbe and Loire. A significantly higher amount of gravel and sand bars within the river bed strongly supported the overall higher morphological status of the Loire. A PCA highlighted that the parameters Wetlands, Active Riparian Zones and Free Flowing Sections similarly described a good morphological situation. In contrast, Canal, Dams and Impervious Surface indicated bad morphological conditions. Finally, the approach was successfully validated with data from a detailed, field-based morphological assessment for the Danube. LaRiMo represents an efficient and cost-effective approach to assess large river morphology across large extents. This method provides comparable results across countries and regions.

Molecular association and morphological characterisation of Himalopsyche larval types (Trichoptera, Rhyacophilidae).

Himalopsyche Banks, 1940 (Trichoptera, Rhyacophilidae) is a genus of caddisflies inhabiting mountain and alpine environments in Central and East Asia and the Nearctic. Of 53 known species, only five species have been described previously in the aquatic larval stage. We perform life stage association using three strategies (GMYC, PTP, and reciprocal monophyly) based on fragments of two molecular markers: the nuclear CAD, and the mitochondrial COI gene. A total of 525 individuals from across the range of Himalopsyche (Himalayas, Hengduan Shan, Tian Shan, South East Asia, Japan, and western North America) was analysed and 32 operational taxonomic units (OTUs) in our dataset delimited. Four distinct larval types of Himalopsyche are uncovered, and these are defined as the phryganea type, japonica type, tibetana type, and gigantea type and a comparative morphological characterisation of the larval types is presented. The larval types differ in a number of traits, most prominently in their gill configuration, as well as in other features such as setal configuration of the pronotum and presence/absence of accessory hooks of the anal prolegs.

Description of the larva of Oecetis mizrain Malicky & Graf, 2012 (Trichoptera, Leptoceridae) and Lepidostoma scotti (Ulmer, 1930) (Trichoptera, Lepidostomatidae) from Chilimo Forest, Central Ethiopia.

The Ethiopian caddisfly fauna comprises 85 species, including 10 Oecetis species and three Lepidostoma species. In this context we provide the first species-level descriptions of Ethiopian caddisfly larvae. We describe and illustrate the larvae of O. mizrain and L. scotti, with additional notes on their habitats and distribution.

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems.

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.

Isoperla vjosae sp. n., a new species of the Isoperla tripartita group from Albania (Plecoptera: Perlodidae).

In this contribution, we describe Isoperla vjosae sp. nov. from Albania. We characterize males, females, and larvae of the new species collected at the Vjosa River using morphological and molecular approaches. Isoperla vjosae sp.nov. is a member of the I. tripartita group, which is widely distributed in the Balkans. This new endemic species is being threatened by a proposed hydroelectric power dam on the Vjosa River impacting the type locality - a large, shifting gravel, low altitude River - an atypical habitat for larvae of the I. tripartita group. For the first time we use molecular tools to delineate Isoperla species from the Balkans.